Semicarbazide-sensitive amine oxidase (SSAO), also known as primary amine oxidase, plasma amine oxidase and benzylamine oxidase, is identical in structure to vascular adhesion protein-1 (VAP-1). In the following discussion, SSAO/VAP-1 is used to describe this protein. The role of this protein in inflammatory diseases has been reviewed (see, for example, Smith D. J. and Vaino P. J., Targeting Vascular Adhesion Protein-1 to Treat Autoimmune and Inflammatory Diseases. Ann. N.Y. Acad. Sci. 2007, 1110, 382-388; and McDonald I. A. et al., Semicarbazide Sensitive Amine Oxidase and Vascular Adhesion Protein-1: One Protein Being Validated as a Therapeutic Target for Inflammatory Diseases. Annual Reports in Medicinal Chemistry, 2008, 43, 229-241).
In most organisms, including humans, two families of mammalian amine oxidases metabolize various mono-, di-, and polyamines produced endogenously or absorbed from exogenous sources. These include the monoamine oxidases (MAO-A and MAO-B) which are present in the mitochondria of most cell types and use covalently bound flavin adenine dinucleotide (FAD) as the cofactor. Polyamine oxidase is another FAD-dependent amine oxidase which oxidatively deaminates spermine and spermidine. SSAO/VAP-1 belongs to the second family which is dependent on copper and uses other co-factors apart from FAD, such as an oxidized tyrosine residue (abbreviated as TPQ or LTQ). MAO and SSAO/VAP-1 oxidatively deaminate some common substrates which includes the monoamines such dopamine, tyramine and benzylamine. SSAO/VAP-1 also oxidizes endogenous methylamine and aminoacetone.
Some of these enzymes were originally defined by the ability of certain compounds to inhibit the enzymatic activity thereof. For example MAO-A is selectively inhibited by clorgyline, MAO-B by L-deprenyl, while neither clorgyline nor L-deprenyl can inhibit the amine oxidase activity of SSAO/VAP-1. SSAO/VAP-1 can be inhibited by semicarbazide, hence the name semicarbazide sensitive amine oxidase.
SSAO/VAP-1 is an ectoenzyme containing a very short cytoplasmic tail, a single transmembrane domain, and a large, highly glycosylated extracellular domain which contains the active center for the amine oxidase activity. SSAO/VAP-1 is also present in a soluble form circulating in the plasma of some animals. It has been shown that this form is a cleaved product of membrane-bound SSAO/VAP-1.
SSAO/VAP-1 appears to have two physiological functions: the first is the amine oxidase activity mentioned above and the second is cell adhesion activity. Both activities are associated with inflammatory processes. SSAO/VAP-1 was shown to play an important role in extravasation of inflammatory cells from the circulation to sites of inflammation (Salmi M. and Jalkanen S., VAP-1: an adhesin and an enzyme. Trends Immunol. 2001, 22, 211-216). VAP-1 antibodies have been demonstrated to attenuate inflammatory processes by blocking the adhesion site of the SSAO/VAP-1 protein and, together with a substantial body of evidence of in vitro and in vivo knockouts, it is now clear that SSAO/VAP-1 is an important cellular mediator of inflammation. Transgenic mice lacking SSAO/VAP-1 show reduced adhesion of leukocytes to endothelial cells, reduced lymphocyte homing to the lymph nodes and a concomitant attenuated inflammatory response in a peritonitis model. These animals were otherwise healthy, grew normally, were fertile, and examination of various organs and tissues showed the normal phenotype. Furthermore, inhibitors of the amine oxidase activity of SSAO/VAP-1 have been found to interfere with leukocyte rolling, adhesion and extravasation and, similar to SSAO/VAP-1 antibodies, exhibit anti-inflammatory properties.
Inflammation is the first response of the immune system to infection or irritation. The migration of leukocytes from the circulation into tissues is essential for this process. Inappropriate inflammatory responses can result in local inflammation of otherwise healthy tissue which can lead to disorders such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis and respiratory diseases. Leukocytes first adhere to the endothelium via binding to adhesion molecules before they can start the process of passing through the walls of the blood vessels. Membrane bound SSAO/VAP-1 is abundantly expressed in vascular endothelial cells such as high venule endothelial cells (HVE) of lymphatic organs and is also expressed in hepatic sinusoidal endothelial cells (HSEC), smooth muscle cells and adipocytes. The expression of SSAO/VAP-1 on the cell surface of endothelial cells is tightly regulated and is increased during inflammation. In the presence of an SSAO/VAP-1 substrate (benzylamine), NFκB was activated in HSECs together with up-regulation of other adhesion molecules, E-selectin and chemokine CXCL8 (IL-8) in vitro. A recent study confirms this result by showing (by mutagenesis) that the transcription and translation of E-selectin and P-selectin is induced by the enzyme activity of SSAO/VAP-1. These results suggest an important role of the amine oxidase activity of SSAO/VAP-1 in the inflammatory response. It has been reported that the oxidase activity of SSAO/VAP-1 induces endothelial E- and P-selectins and leukocyte binding (Jalkanen, S. et al., The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and P-selectins and leukocyte binding. Blood 2007, 110, 1864-1870).
Excessive and chronic inflammatory responses have been associated with the symptoms of many chronic diseases, such as rheumatoid arthritis, multiple sclerosis, asthma and chronic obstructive pulmonary disease (COPD). Patients suffering from either atopic eczema or psoriasis (both chronic inflammatory skin disorders) have higher levels of SSAO/VAP-1 positive cells in their skin compared to skin from healthy controls.
Asthma can be considered a disease resulting from chronic inflammation of the airways which results in bronchoconstriction and excessive build-up of mucus. Many patients can be adequately treated with bronchodilators (eg, β2 agonists, leukotriene antagonists and with inhaled steroids). However, up to about 20% of patients suffer from severe asthma and don't respond well to these treatments. A subset of these patients are resistant to inhaled steroids and present with high neutrophil counts in their lung fluids. SSAO/VAP-1 is expressed in the lungs and plays a role in the trafficking of neutrophils.
Another subset of asthma patients is acutely sensitive to viral infections of the airways; such infections exacerbate the underlying inflammation and can lead to severe asthma attacks.
It has been recently recognized that patients suffering from cystic fibrosis frequently suffer from persistent lung inflammation which can be independent from chronic lung infection. It has been argued that tissue damage in cystic fibrosis patients is due to mediators released by neutrophils. While standard antibiotic treatment to clear bacterial infection would be expected to resolve the underlying inflammation if the inflammation were solely due to the infection, data from recent studies demonstrate that this is not the case and that the airways are in a neutrophil-driven pro-inflammatory state primed for excessive and prolonged inflammatory response to bacterial infection. See Rao S. and Grigg J., New insights into pulmonary inflammation in cystic fibrosis. Arch Dis Child 2006, 91:786-788.
SSAO/VAP-1 is also highly expressed in adipocytes where it plays a role in glucose transport independent of the presence of insulin. It has been observed that levels of plasma SSAO/VAP-1 are increased in patients suffering from diabetes. Elevated levels of plasma SSAO/VAP-1 have been found in patients suffering from other illnesses, such as congestive heart failure and liver cirrhosis. It has been suggested that SSAO/VAP-1 is associated with most, if not all, inflammatory diseases whether the inflammation is in response to an immune response or subsequent to other events such as occlusion and reperfusion of blood vessels.
It has been recognized in recent years that SSAO/VAP-1 is expressed in sinusoidal endothelial cells in the liver and that this protein is believed to be associated with hepatic disease, in particular liver fibrosis (Weston C. J. and Adams D. H., Hepatic consequences of vascular adhesion protein-1 expression, J Neural Transm 2011; 118:1055-1064). Furthermore, a VAP-1 antibody and a small molecule inhibitor were found to attenuate carbon tetrachloride induced fibrosis in mice. Thus, SSAO/VAP-1 inhibitors have the potential to treat fibrotic disease (WO 2011/029996). It has been recently reported that oxidation of methylamine by SSAO/VAP-1 in the presence of tumor necrosis factor α induces the expression of MAdCAM-1 in hepatic vessels, and that this is associated with the hepatic complications of inflammatory bowel disease (IBD) (Liaskou W. et al., Regulation of Mucosal Addressin Cell Adhesion Molecule 1 Expression in Human and Mice by Vascular Adhesion Protein 1 Amine Oxidase Activity, Hepatology 2011; 53, 661-672).
It has been reported that SSAO/VAP-1 inhibitors can attenuate angiogenesis and lymphangiogenesis, and that these inhibitors offer potential to treat ocular diseases such as macular degeneration, corneal angiogenesis, cataracts, and inflammatory conditions such as uveitis (US 2009/0170770; WO 2009/051223; Noda K., et al., Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis, FASEB J. 2008, 22, 1094-1103).
Increased levels of SSAO/VAP-1 were observed in the serum of patients suffering from hepatocellular carcinoma. In a murine melanoma model, small molecule SSAO/VAP-1 inhibitors were shown to retard tumor growth, in contrast to VAP-1 antibodies which had no activity (Weston C. J. and Adams D. H., Hepatic consequences of vascular adhesion protein-1 expression, J Neural Transm 2011, 118, 1055-1064).
It was reported that SSAO/VAP-1 plays an important role in cancer biology (Marttila-Ichihara F. et al. Small-Molecule Inhibitors of Vascular Adhesion Protein-1 Reduce the Accumulation of Myeloid Cells into Tumors and Attenuate Tumor Growth in Mice. The Journal of Immunology, 2010, 184, 3164-3173). SSAO/VAP-1 small molecule inhibitors reduced the number of proangiogenic Gr-1+CD11b+ myeloid cells in melanomas and lymphomas.
During the SSAO/VAP-1 amine oxidase catalytic cycle the covalently bound cofactor, TPQ, is first reduced, and then re-oxidized by oxygen in the presence of copper with the generation of hydrogen peroxide as a by-product. It has been speculated that excessive hydrogen peroxide concentrations can be deleterious and may contribute to the pathology of various inflammatory and neurodegenerative processes (Götz M. E., et al., Oxidative stress: Free radical production in neural degeneration. Pharmacol Ther 1994, 63, 37-122).
Inflammation is believed to be an important feature of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis, and similarly is a feature of the pathophysiology that occurs after a cerebral occlusion/reperfusion event (Aktas, O. et al., Neuronal damage in brain inflammation. Arch Neurol 2007, 64, 185-9). Excessive activity SSAO/VAP-1 has been independently implicated in these processes (Xu, H-L., et al., Vascular Adhesion Protein-1 plays an important role in postischemic inflammation and neuropathology in diabetic, estrogen-treated ovariectomized female rats subjected to transient forebrain ischemia. Journal Pharmacology and Experimental Therapeutics, 2006, 317, 19-26).
Some known MAO inhibitors also inhibit SSAO/VAP-1 (e.g., the MAO-B inhibitor Mofegiline illustrated below). Mofegiline has been reported to inhibit experimental autoimmune encephalomyelitis (US 2006/0025438 A1). This inhibitor is a member of the haloallylamine family of MAO inhibitors; the halogen in Mofegiline is fluorine. Fluoroallylamine inhibitors are described in U.S. Pat. No. 4,454,158. There have been reports of a chloroallylamine, MDL72274 (illustrated below), selectively inhibiting rat SSAO/VAP-1 compared to MAO-A and MAO-B:

Additional fluoroallylamine inhibitors are described in U.S. Pat. No. 4,699,928; the two compounds illustrated below were described as selective inhibitors of MAO-B:

Other examples structurally related to Mofegiline can be found in WO 2007/120528.
Haloallylamine compounds that differ from Mofegiline in core structure have been synthesized and were shown to inhibit the amine oxidase activity from copper-dependent amine oxidases from a number of species (see Kim J., et al., Inactivation of bovine plasma amine oxidase by haloallylamines. Bioorg Med Chem 2006, 14, 1444-1453). These compounds have been included in a patent application (WO 2007/005737):

WO 2009/066152 describes a family of 3-substituted 3-haloallylamines that are inhibitors of SSAO/VAP-1 and are claimed as treatment for a variety of indications, including inflammatory disease. The following compounds are specifically described:

References to the effects of SSAO/VAP-1 inhibitors in various animal models of disease can be found in the review publication by McDonald I. A. et al., Semicarbazide Sensitive Amine Oxidase and Vascular Adhesion Protein-1: One Protein Being Validated as a Therapeutic Target for Inflammatory Diseases. Annual Reports in Medicinal Chemistry, 2008, 43, 229-241 and in the following publications, O'Rourke A. M. et al., Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity. J. Pharmacol. Exp. Ther., 2008, 324, 867-875; and O'Rourke A. M. et al., Benefit of inhibiting SSAO in relapsing experimental encephalomyelitis. J. Neural. Transm., 2007, 114, 845-849.